Saccharomyces Cerevisiae Plasma Membrane Research Articles

Let it stick: Strategies and applications for intracellular plasma membrane targeting of proteins in Saccharomyces cerevisiae.

Lipid binding domains and protein lipidations are essential features to recruit proteins to intracellular membranes, enabling them to function at specific sites within the cell. Membrane association can also be exploited to answer fundamental and applied research questions, from obtaining insights into the understanding of lipid metabolism to employing them for metabolic engineering to redirect fluxes. This review presents a broad catalog of membrane binding strategies focusing on the plasma membrane of Saccharomyces cerevisiae. Both lipid binding domains (pleckstrin homology, discoidin-type C2, kinase associated-1, basic-rich and bacterial phosphoinositide-binding domains) and co- and post-translational lipidations (prenylation, myristoylation and palmitoylation) are introduced as tools to target the plasma membrane. To provide a toolset of membrane targeting modules, respective candidates that facilitate plasma membrane targeting are showcased including their in vitro and in vivo properties. The relevance and versatility of plasma membrane targeting modules are further highlighted by presenting a selected set of use cases.

The phosphatase Glc7 controls the eisosomal response to starvation via post-translational modification of Pil1.

The yeast (Saccharomyces cerevisiae) plasma membrane (PM) is organised into specific subdomains that regulate surface membrane proteins. Surface transporters actively uptake nutrients in particular regions of the PM where they are also susceptible to substrate-induced endocytosis. However, transporters also diffuse into distinct subdomains termed eisosomes, where they are protected from endocytosis. Although most nutrient transporter populations are downregulated in the vacuole following glucose starvation, a small pool is retained in eisosomes to provide efficient recovery from starvation. We find the core eisosome subunit Pil1, a Bin, Amphiphysin and Rvs (BAR) domain protein required for eisosome biogenesis, is phosphorylated primarily by the kinase Pkh2. In response to acute glucose starvation, Pil1 is rapidly dephosphorylated. Enzyme localisation and activity screens suggest that the phosphatase Glc7 is the primary enzyme responsible for Pil1 dephosphorylation. Defects in Pil1 phosphorylation, achieved by depletion of GLC7 or expression of phospho-ablative or phospho-mimetic mutants, correlate with reduced retention of transporters in eisosomes and inefficient starvation recovery. We propose that precise post-translational control of Pil1 modulates nutrient transporter retention within eisosomes, depending on extracellular nutrient levels, to maximise recovery following starvation.

Lipid flippases in polarized growth.

Polarized growth is required in eukaryotic cells for processes such as cell division, morphogenesis and motility, which involve conserved and interconnected signalling pathways controlling cell cycle progression, cytoskeleton reorganization and secretory pathway functioning. While many of the factors involved in polarized growth are known, it is not yet clear how they are coordinated both spatially and temporally. Several lines of evidence point to the important role of lipid flippases in polarized growth events. Lipid flippases, which mainly belong to the P4 subfamily of P-type ATPases, are active transporters that move different lipids to the cytosolic side of biological membranes at the expense of ATP. The involvement of the Saccharomyces cerevisiae plasma membrane P4 ATPases Dnf1p and Dnf2p in polarized growth and their activation by kinase phosphorylation were established some years ago. However, these two proteins do not seem to be responsible for the phosphatidylserine internalization required for early recruitment of proteins to the plasma membrane during yeast mating and budding. In a recent publication, we demonstrated that the Golgi-localized P4 ATPase Dnf3p has a preference for PS as a substrate, can reach the plasma membrane in a cell cycle-dependent manner, and is regulated by the same kinases that activate Dnf1p and Dnf2p. This finding solves a long-lasting enigma in the field of lipid flippases and suggests that tight and heavily coordinated spatiotemporal control of lipid translocation at the plasma membrane is important for proper polarized growth.

Functional Significance of Conserved Cysteines in the Extracellular Loops of the ATP Binding Cassette Transporter Pdr11p.

The pleiotropic drug resistance (PDR) transporter Pdr11p is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae, where it facilitates the uptake of exogenous sterols. Members of the fungal PDR family contain six conserved cysteines in their extracellular loops (ECL). For the functional analysis of these cysteine residues in Pdr11p, we generated a series of single cysteine-to-serine mutants. All mutant proteins expressed well and displayed robust ATPase activity upon purification. Mass-spectrometry analysis identified two cysteine residues (C582 and C603) in ECL3 forming a disulfide bond. Further characterization by cell-based assays showed that all mutants are compromised in facilitating sterol uptake, protein stability, and trafficking to the plasma membrane. Our data highlight the fundamental importance of all six extracellular cysteine residues for the functional integrity of Pdr11p and provide new structural insights into the PDR family of transporters.

Point mutations in the different domains of the Saccharomyces cerevisiae plasma membrane PMA1 ATPase cause redistribution among fractions of inorganic polyphosphates

Both ATP and inorganic polyphosphates (PolyP) appeared to be involved in the yeast energy homeostasis, in which plasma membrane PMA1 H+-АТРase plays one of the key roles. During biogenesis and functioning, the enzyme undergoes structural and regulatory phosphorylation. Aim of the work was to elucidate interconnection between functioning of the yeast PMA1 H+-АТРase carrying point substitutions that affected the enzyme structure-function relationship and its ability to be phosphorylated and PolyP metabolism. Effect of such replacements of phosphorylable and non-phosphorylable residues in three topologically and functionally different domains of the enzyme – membrane, extracytosolic, and C-terminal – on the metabolism of polyphosphates and distribution between short-, mid-, and long-chained PolyP fractions (PolyP1-PolyP4-5) has been studied. АТРase activity of membrane and most extracytosolic strains was noticeably lower comparing to the wild type. Of these mutants, three substitutions (L801A, E803A, E847A) have not caused significant changes in PolyP content regardless up to twofold drop of the ATPase activity; F796A with four-fold decreased activity has led to noticeable increase of mid-chained PolyP fractions. The most pronounced effect of PolyP redistribution was caused either by removal of potential (S846A, T850A, D851A) or established (S911A) phosphosites in the PMA1 ATPase or by altering type of the established phosphosite (S911D, T912D). Patterns of PolyP fractions for these two groups have significantly differed from each other, occurring in opposite directions for mutants with removed and changed phosphosite. Changing residue of phosphosite without altering its type (T850S) has not led to significant changes in PolyP content. Communicated by Ramaswamy H. Sarma

Weak Acid Permeation in Synthetic Lipid Vesicles and Across the Yeast Plasma Membrane

We present a fluorescence-based approach for determination of the permeability of small molecules across the membranes of lipid vesicles and living cells. With properly designed experiments, the method allows us to assess the membrane physical properties both in vitro and in vivo. We find that the permeability of weak acids increases in the order of benzoic > acetic > formic > lactic, both in synthetic lipid vesicles and the plasma membrane of Saccharomyces cerevisiae, but the permeability is much lower in yeast (one to two orders of magnitude). We observe a relation between the molecule permeability and the saturation of the lipid acyl chain (i.e., lipid packing) in the synthetic lipid vesicles. By analyzing wild-type yeast and a manifold knockout strain lacking all putative lactic acid transporters, we conclude that the yeast plasma membrane is impermeable to lactic acid on timescales up to ∼2.5 h.

Proton-solute coupling mechanism of the maltose transporter from Saccharomyces cerevisiae

Mal11 catalyzes proton-coupled maltose transport across the plasma membrane of Saccharomyces cerevisiae. We used structure-based design of mutants and a kinetic analysis of maltose transport to determine the energy coupling mechanism of transport. We find that wildtype Mal11 is extremely well coupled and allows yeast to rapidly accumulate maltose to dangerous levels, resulting under some conditions in self-lysis. Three protonatable residues lining the central membrane-embedded cavity of Mal11 were identified as having potential roles in proton translocation. We probed the mechanistic basis for proton coupling with uphill and downhill transport assays and found that single mutants can still accumulate maltose but with a lower coupling efficiency than the wildtype. Next, we combined the individual mutations and created double and triple mutants. We found some redundancy in the functions of the acidic residues in proton coupling and that no single residue is most critical for proton coupling to maltose uptake, unlike what is usually observed in related transporters. Importantly, the triple mutants were completely uncoupled but still fully active in downhill efflux and equilibrium exchange. Together, these results depict a concerted mechanism of proton transport in Mal11 involving multiple charged residues.

Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p.

The ATP binding cassette (ABC) transporters Pdr11p and its paralog Aus1p are expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and are required for sterol uptake. However, the precise mechanism by which these ABC transporters facilitate sterol movement is unknown. In this study, an overexpression and purification procedure was developed with the aim to characterise the Pdr11p transporter. Engineering of Pdr11p variants fused at the C terminus with green fluorescent protein (Pdr11p-GFP) and containing a FLAG tag at the N terminus facilitated expression analysis and one-step purification, respectively. The detergent-solubilised and purified protein displayed a stable ATPase activity with a broad pH optimum near 7.4. Mutagenesis of the conserved lysine to methionine (K788M) in the Walker A motif abolished ATP hydrolysis. Remarkably, and in contrast to Aus1p, ATPase activity of Pdr11p was insensitive to orthovanadate and not specifically stimulated by phosphatidylserine upon reconstitution into liposomes. Our results highlight distinct differences between Pdr11p and Aus1p and create an experimental basis for further biochemical studies of both ABC transporters to elucidate their function.

Heterologous expression of the yeast Tpo1p or Pdr5p membrane transporters in Arabidopsis confers plant xenobiotic tolerance

Soil contamination is a major hindrance for plant growth and development. The lack of effective strategies to remove chemicals released into the environment has raised the need to increase plant resilience to soil pollutants. Here, we investigated the ability of two Saccharomyces cerevisiae plasma-membrane transporters, the Major Facilitator Superfamily (MFS) member Tpo1p and the ATP-Binding Cassette (ABC) protein Pdr5p, to confer Multiple Drug Resistance (MDR) in Arabidopsis thaliana. Transgenic plants expressing either of the yeast transporters were undistinguishable from the wild type under control conditions, but displayed tolerance when challenged with the herbicides 2,4-D and barban. Plants expressing ScTPO1 were also more resistant to the herbicides alachlor and metolachlor as well as to the fungicide mancozeb and the Co2+, Cu2+, Ni2+, Al3+ and Cd2+ cations, while ScPDR5-expressing plants exhibited tolerance to cycloheximide. Yeast mutants lacking Tpo1p or Pdr5p showed increased sensitivity to most of the agents tested in plants. Our results demonstrate that the S. cerevisiae Tpo1p and Pdr5p transporters are able to mediate resistance to a broad range of compounds of agricultural interest in yeast as well as in Arabidopsis, underscoring their potential in future biotechnological applications.

The Salmonella effector SteA binds phosphatidylinositol 4-phosphate for subcellular targeting within host cells.

Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4-phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella-containing vacuole (SCV) and to Salmonella-induced tubules; using the PI(4)P-binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N-terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells.

Changes in the Sterol Composition of the Plasma Membrane Affect Membrane Potential, Salt Tolerance and the Activity of Multidrug Resistance Pumps in Saccharomyces cerevisiae.

We investigated the impact of the deletions of genes from the final steps in the biosynthesis of ergosterol (ERG6, ERG2, ERG3, ERG5, ERG4) on the physiological function of the Saccharomyces cerevisiae plasma membrane by a combination of biological tests and the diS-C3(3) fluorescence assay. Most of the erg mutants were more sensitive than the wild type to salt stress or cationic drugs, their susceptibilities were proportional to the hyperpolarization of their plasma membranes. The different sterol composition of the plasma membrane played an important role in the short-term and long-term processes that accompanied the exposure of erg strains to a hyperosmotic stress (effect on cell size, pH homeostasis and survival of yeasts), as well as in the resistance of cells to antifungal drugs. The pleiotropic drug-sensitive phenotypes of erg strains were, to a large extent, a result of the reduced efficiency of the Pdr5 efflux pump, which was shown to be more sensitive to the sterol content of the plasma membrane than Snq2p. In summary, the erg4Δ and erg6Δ mutants exhibited the most compromised phenotypes. As Erg6p is not involved in the cholesterol biosynthetic pathway, it may become a target for a new generation of antifungal drugs.

Specific phosphoantibodies reveal two phosphorylation sites in yeast Pma1 in response to glucose.

Glucose triggers post-translational modifications of the Saccharomyces cerevisiae plasma membrane H(+)-ATPase (Pma1) that lead to an increase in enzyme activity. The activation results from changes in two kinetic parameters: an increase in the affinity of the enzyme for ATP, depending on Ser899, and an increase in the Vmax involving Ser911/Thr912. Using phosphospecific antibodies, we show that Ser899 and Ser911/Thr912 are phosphorylated in vivo during glucose activation and that protein phosphatase Glc7 is involved in the dephosphorylation of Ser899 upon glucose starvation.

Kch1 Family Proteins Mediate Essential Responses to Endoplasmic Reticulum Stresses in the Yeasts Saccharomyces cerevisiae and Candida albicans

The activation of a high affinity Ca(2+) influx system (HACS) in the plasma membrane is required for survival of yeast cells exposed to natural or synthetic inhibitors of essential processes (secretory protein folding or sterol biosynthesis) in the endoplasmic reticulum (ER). The mechanisms linking ER stress to HACS activation are not known. Here we show that Kch1, a recently identified low affinity K(+) transporter in the plasma membrane of Saccharomyces cerevisiae, is up-regulated in response to several ER stressors and necessary for HACS activation. The activation of HACS required extracellular K(+) and was also dependent on the high affinity K(+) transporters Trk1 and Trk2. However, a paralog of Kch1 termed Kch2 was not expressed and not necessary for HACS activation in these conditions. The pathogenic yeast Candida albicans carries only one homolog of Kch1/Kch2, and homozygous knock-out mutants were similarly deficient in the activation of HACS during the responses to tunicamycin. However, the Kch1 homolog was not necessary for HACS activation or cell survival in response to several clinical antifungals (azoles, allylamines, echinocandins) that target the ER or cell wall. Thus, Kch1 family proteins represent a conserved linkage between HACS and only certain classes of ER stress in these yeasts.

Characterization of Ca2+/H+ exchange in the plasma membrane of Saccharomyces cerevisiae

The characteristics of the Ca2+/H+ exchange were directly investigated in functionally inverted (inside-out) plasma membrane vesicles isolated from yeast using an aqueous two-phase partitioning method. Results showed that following the generation of an inside-acid pH gradient (fluorescence quenching), addition of Ca2+ caused movement of H+ out of the vesicles (fluorescence recovery). The Ca2+/H+ exchange displayed saturation kinetics with respect to extravesicular Ca2+ and ATP concentrations in the plasma membrane, and showed specificity for Ca2+. The protonophore FCCP (carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone), abolished the fluorescence quenching and consequently inhibited Ca2+/H+ exchange in plasma membrane vesicles. Vanadate, which is known to inhibit the plasma membrane H+-ATPase, significantly decreased the Ca2+-dependent transport of H+ out of vesicles. When the electrical potential across the plasma membrane was dissipated with valinomycin and potassium, the rate of Ca2+/H+ exchange increased compared to that of the control without valinomycin, indicating that the stoichiometric ratio for this exchange is greater than 2H+:Ca2+. These data suggest that Ca2+ is transported out of yeast cells through a Ca2+/H+ exchange system that is driven by the proton-motive force generated by the plasma membrane H+-ATPase.

The basidiomycete Ustilago maydis has two plasma membrane H+-ATPases related to fungi and plants

The fungal and plant plasma membrane H⁺-ATPases play critical roles in the physiology of yeast, plant and protozoa cells. We identified two genes encoding two plasma membrane H⁺-ATPases in the basidiomycete Ustilago maydis, one protein with higher identity to fungal (um02581) and the other to plant (um01205) H⁺-ATPases. Proton pumping activity was 5-fold higher when cells were grown in minimal medium with ethanol compared to cells cultured in rich YPD medium, but total vanadate-sensitive ATPase activity was the same in both conditions. In contrast, the activity in cells cultured in minimal medium with glucose was 2-fold higher than in YPD or ethanol, implicating mechanisms for the regulation of the plasma membrane ATPase activity in U. maydis. Analysis of gene expression of the H⁺-ATPases from cells grown under different conditions, showed that the transcript expression of um01205 (plant-type) was higher than that of um02581 (fungal-type). The translation of the two proteins was confirmed by mass spectrometry analysis. Unlike baker's yeast and plant H⁺-ATPases, where the activity is increased by a short incubation with glucose or sucrose, respectively, U. maydis H⁺-ATPase activity did not change in response to these sugars. Sequence analysis of the two U. maydis H⁺-ATPases revealed the lack of canonical threonine and serine residues which are targets of protein kinases in Saccharomyces cerevisiae and Arabidopsis thaliana plasma membrane H⁺-ATPases, suggesting that phosphorylation of the U. maydis enzymes occurs at different amino acid residues.

Superoxide Triggers an Acid Burst in Saccharomyces cerevisiae to Condition the Environment of Glucose-starved Cells

Although yeast cells grown in abundant glucose tend to acidify their extracellular environment, they raise the pH of the environment when starved for glucose or when grown strictly with non-fermentable carbon sources. Following prolonged periods in this alkaline phase, Saccharomyces cerevisiae cells will switch to producing acid. The mechanisms and rationale for this "acid burst" were unknown. Herein we provide strong evidence for the role of mitochondrial superoxide in initiating the acid burst. Yeast mutants lacking the mitochondrial matrix superoxide dismutase (SOD2) enzyme, but not the cytosolic Cu,Zn-SOD1 enzyme, exhibited marked acceleration in production of acid on non-fermentable carbon sources. Acid production is also dramatically enhanced by the superoxide-producing agent, paraquat. Conversely, the acid burst is eliminated by boosting cellular levels of Mn-antioxidant mimics of SOD. We demonstrate that the acid burst is dependent on the mitochondrial aldehyde dehydrogenase Ald4p. Our data are consistent with a model in which mitochondrial superoxide damage to Fe-S enzymes in the tricarboxylic acid (TCA) cycle leads to acetate buildup by Ald4p. The resultant expulsion of acetate into the extracellular environment can provide a new carbon source to glucose-starved cells and enhance growth of yeast. By triggering production of organic acids, mitochondrial superoxide has the potential to promote cell population growth under nutrient depravation stress.

Activation of H+-ATPase of the Plasma Membrane of Saccharomyces cerevisiae by Glucose: The Role of Sphingolipid and Lateral Enzyme Mobility

Activation of the plasma membrane H+-ATPase of the yeast Saccharomyces cerevisiae by glucose is a complex process that has not yet been completely elucidated. This study aimed to shed light on the role of lipids and the lateral mobility of the enzyme complex during its activation by glucose. The significance of H+-ATPase oligomerization for the activation of H+-ATPase by glucose was shown using the strains lcb1-100 and erg6, with the disturbed synthesis of sphyngolipid and ergosterol, respectively. Experiments with GFP-fused H+-ATPase showed a decrease in fluorescence anisotropy during the course of glucose activation, suggesting structural reorganization of the molecular domains. An immunogold assay showed that the incubation with glucose results in the spatial redistribution of ATPase complexes in the plasma membrane. The data suggest that (1) to be activated by glucose, H+-ATPase is supposed to be in an oligomeric state, and (2) glucose activation is accompanied by the spatial movements of H+-ATPase clusters in the PM.

Sterol Properties Required for Microdomain Formation: From Model Systems to Living Yeast and Mammalian Cells

This work presents new insights into the evolution of sterol-enriched domains in the plasma membrane (PM) of eukaryotes. The recent finding of sphingolipid-enriched gel domains in the PM of Saccharomyces cerevisiae(1) demands novel strategies to study sterol-enriched liquid ordered domains in this organism. Therefore, we employed di-4-ANEPPS fluorescence, here shown to be sensitive to S. cerevisiae sterols and Fluorescence Lifetime Imaging Microscopy (FLIM) for the first time in S. cerevisiae PM. In vivo studies were performed with S. cerevisiae (wild type and erg6Δ), and animal (CHO) cells, modeled by liposomes containing ergosterol, zymosterol, or cholesterol, respectively. Different fluorescent probes were used, showing that these sterols had comparable effects regarding lipid bilayer order and complete solubilization of gel domains. However, confocal fluorescence microscopy of Giant Unilamellar Vesicles showed that zymosterol, a biosynthetic precursor of ergosterol and cholesterol, has no ability to form lipid rafts.In living cells, sterol-induced emission blue-shift and longer fluorescence lifetime of di-4-ANEPPS increased as: wild type<erg6Δ<CHO. Concomitantly, in model systems, cholesterol and zymosterol had much larger effects than ergosterol in di-4-ANEPPS fluorescence. However, zymosterol was unique because it yielded overlapping results for both saturated and unsaturated lipids. The two other sterols produced much stronger alterations with a saturated lipid, even though the absolute effect in di-4-ANEPPS fluorescence was often smaller than that of zymosterol. Hence, the differential regarding the interaction of sterols with saturated versus unsaturated lipids emerges as a requisite for lipid raft formation and as an important factor in sterol evolution.(1) F. Aresta-Branco, A.M. Cordeiro, H.S. Marinho, L. Cyrne, F. Antunes, R.F.M. de Almeida (2011) “Gel domains in the plasma membrane of Saccharomyces cerevisiae: highly ordered, ergosterol-free, sphingolipid-enriched lipid rafts.” J. Biol. Chem. 286:5043-5054.Acknowledgements: PTDC/QUI-BIQ/104311/2008 and Ciencia2007 (FCT, Portugal).

Sphingolipid-Enriched Microdomains in the Plasma Membrane of Saccharomyces Cerevisiae: Ergosterol-Free «Lipid Rafts» in the Gel Phase

The plasma membrane (PM) of Saccharomyces cerevisiae was studied using the probes trans-parinaric acid (t-PnA) and diphenylhexatriene (DPH), providing the first direct evidence for the presence of gel domains in living cells and showing that the PM is mostly constituted by ordered domains(1). The fluorescence lifetimes of t-PnA are particularly sensitive to the presence and nature of ordered domains, and here the first measurements in yeast cells are reported. A long fluorescence lifetime typical of the gel phase (> 30 ns) was found in mid-exponential phase wild-type (wt) cells from two different genetic backgrounds, at both 24 and 30°C. The confirmation of the gel nature was achieved by comparing t-PnA and DPH fluorescence anisotropy, and studying the thermotropic transitions of liposomes reconstituted from PM lipid-extracts. To address the location and composition of the domains spheroplasts, the isolated PM, liposomes from total lipid-extracts, and living cells of several mutant strains with deletions in genes coding for enzymes involved in ergosterol-, sphingolipid- and GPI-anchor biosynthesis were also studied. It was shown that the gel domains are not ergosterol-enriched lipid rafts, but mainly composed of sphingolipids and GPI-anchored proteins, suggesting important roles in membrane traffic, signaling, and interactions with the cell wall. The findings provide a biophysical mechanism for the targeting of some proteins to the PM, which is sphingolipid-dependent but sterol-independent and for the formation of membrane compartments in the yeast PM (MCP and MCC)(2). Acknowledgements: PTDC/QUI-BIQ/104311/2008, Ciência2007 and PEst-OE/QUI/UI0612/2011 (FCT, Portugal). (1) F. Aresta-Branco, A.M. Cordeiro, H.S. Marinho, L. Cyrne, F. Antunes, R.F.M. de Almeida (2011) J. Biol. Chem. 286:5043-5054. (2) Poolman, B. (2011) Evaluation of: [Aresta-Branco F et al. Gel domains in the plasma membrane ...]. Faculty of 1000, 26 Apr 2011. F1000.com/9922956.

Arg 901 in the AE1 C-terminal tail is involved in conformational change but not in substrate binding

In our previous paper, we demonstrated that Arg 901 in the C-terminal tail of human AE1 (band 3, anion exchanger 1) had a functional role in conformational change during anion exchange. To further examine how Arg 901 is involved in conformational change, we expressed various Arg 901 mutants and alanine mutants of the C-terminal tail (from Leu 886 to Val 911) on the plasma membrane of Saccharomyces cerevisiae and evaluated the kinetic parameters of sulfate ion transport. As a result, Vmax decreased as the hydrophobicities of the 901st and peripheral hydrophilic residues increased, indicating that the hydrophobicity of the C-terminal residue is involved in the conformational change. We also found the alkali and protease resistance of the C-terminal region after Arg 901 modification with hydroxyphenylglyoxal (HPG) or phenylglyoxal (PG), a hydrophobic reagent. These results suggested that the increased hydrophobicity of the C-terminal region around Arg 901 leads to inefficient conformational change by the newly produced hydrophobic interaction.